Nanotechnology for Intracellular Nucleic Acid Delivery
Prof. Ke Zhang
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Nanotechnology for Intracellular Nucleic Acid Delivery
Prof. Ke ZhangDepartment of Chemistry and Chemical Biology
Northeastern University, Boston, MA
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Diagnosis and detection
DNA microarraysPCR Bio-barcode assays
Verigene®
Nucleic acids – a biomedical revolution
RNA interference and antisense technologies
Therapeutics
Diagnosis and detection
DNA microarraysPCR Bio-barcode assays
Verigene®
Surprisingly, there are only a handful of therapeutics on the market…
CGGUAACGU
GCCAUUGCA
CGGUAACGU
GCCAGUGCA
CGGUCACGU
GCCAGUGCA
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Nucleic acids as therapeutics
Negative charge
Physiochemical properties
Large!
Body clearance by kidney
Rapid nuclease degradation
Scavenger receptor capture
and intracellular degradation
Nanotechnology for Intracellular Nucleic Acid Delivery
Prof. Ke Zhang
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Zamecnik et al., PNAS, 1978
Higashi et al.,J. Biochem., 1984
VA VV VAV1970 1980 1990 2000 2010
Synthetics
Nucleic acid delivery systems
Naked DNA Viral vectors
Peptides
Astriab-Fisher et al., Biochem.
Pharmacol., 1997
Liposomes
Wong et al.,Gene, 1980
Dendrimers
Haensler et al. Bioconjugate Chem., 1993
Laemmli et al., PNAS 1975
Rosi et al., Science,2006
Polymers Nano
Grillot-Courvalin et al.,Nat. Nanotechnol., 1998
Bacterial vectors
Biologics
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Polymer chain Micelles
HydrophilicBlock
HydrophobicBlock
Self-assembly of amphiphilic block copolymers
Micelles
Block copolymer micelles
200 nm
Spheres
Block segment ratio → Interfacial curvature → Morphology
100 nm
Higher curvature
Lower curvature
0.2 m
Polymer chain
HydrophilicBlock
HydrophobicBlock
Self-assembly of amphiphilic block copolymers
Intra-NP forces
HelicesCylinders Discs Toroids
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Nucleosome-mimicking polymer particles
Shell-crosslinkable• Positively charged
• Around 10 nm in diameter
Design principles:
O O O
Br
NH
NR1R2
O NH
NR1R2
O NH
NR1R2
O NH
NR1R2
O NH
NR1R2
O NH
NR1R2
O NH
NR1R2
O OO
Br
NH
NR1R2
O NH
NR1R2
O NH
NR1R2
O NH
NR1R2
O NH
NR1R2
O NH
NR1R2
O NH
NR1R2
OO
O
Br
NH
NR1R2
ONH
NR1R2
ONH
NR1R2
ONH
NR1R2
ONH
NR1R2
ONH
NR1R2
ONH
NR1R2
O O O
R
NH
NR1 R2
R1, R2 =H or alkyl
Self-assemble
poly(acrylamidoethylamine)-b-polystyrene
Nanotechnology for Intracellular Nucleic Acid Delivery
Prof. Ke Zhang
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7
Cationic nanoparticles transfect cells
Cationic shell-xlinkedknedel-like nanoparticles
(cSCKs)
50 nm
Polyfect cSCK
EGFP transfection in HeLa cells
Lipo
fect
Polyf
ect
2:1
4:1
6:1
8:1
10:1
20:1
0
10
20
30
40
50
% c
ell t
ran
sfe
cte
d
N/P ratio
cSCK
Biomaterials, 2009
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The amine-rich shell of cSCKs
carries dual functions
Exposed amines –DNA complexation
Sterically hindered –Proton absorption
Why does it work, and how can one improve?
The amine-rich shell of cSCKs
carries dual functions
Exposed amines –DNA complexation
Sterically hindered –Proton absorption
vs.
N/P = 1N/P = 2
cSCK Homopolymer Cell0
150
300
450
600
750
900
Flu
ore
sc
en
ce (a.u
.)
N/P=5
N/P=8
N/P=10
N/P=12
N/P=15
N/P=20
N/P=25
cell
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Variation of cSCK shell composition
cSCK-pa100 cSCK-pa50-ta50 cSCK-ta100
cSCK-pa50-ca50 cSCK-ta50-ca50
Increasing tertiary amine
Incre
asin
g c
arb
oxylic a
cid
These changes allow for independent variation
in buffering capacity and binding affinity
Nanotechnology for Intracellular Nucleic Acid Delivery
Prof. Ke Zhang
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Modulating binding affinity/buffering capacity
cSCK-pa100 cSCK-pa50-ta50 cSCK-ta100
cSCK-pa50-ca50 cSCK-ta50-ca50
N/P = 2 N/P = 8
N/P > 32 N/P > 32
N/P = 4
N/P = 8 N/P = 32
Decreasingbufferingcapacity
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Polyf
ect
Lipo
fect
amin
eCel
l
cSCK-p
a 100
cSCK
-pa 75
-ta25
cSCK
-pa 50
-ta50
cSCK
-pa 25
-ta75
cSCK
-ta10
0
0
200
400
600
800
1000
1200
Mean
flu
ore
scen
ce (
a.u
.)
Controls
N/P = 4
N/P = 6
N/P = 10
N/P = 20
N/P = 30
N/P = 40
Improved transfection efficiency
Reduction of binding with DNA
improves transfection efficiency
Transfection in HeLa cells
Biomaterials, 2010; J. Nuc. Acids., 2012; Nanomedicine NBM, 2013;
Nuc. Acid Ther., 2013; Org. Biomol. Chem., 2013
Polyfect Lipofectamine2k
cSCK-pa100 cSCK-pa25ta75
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Oligonucleotide delivery with cSCKs
Luciferase pre-mRNA splicing correction
assay using HeLa pLuc 705 cells
Antisense (ON705)
Luc iferase Luciferase
Defective luciferase
Active luciferase
Kang, S. H.; Cho, M. J.; Kole R.
Biochemistry 1998, 37, 6235
Luc iferase3’ 5’
Pre-mRNA
mRNA
Olig
ofect
amin
e
Polyf
ect
Unt
reat
ed
cSCK-p
a 100
cSCK-p
a 75-ta
25
cSCK-p
a 50-ta
50
cSCK-p
a 25-ta
75
cSCK-ta
100
cSCK-p
a 70-c
a 30
cSCK-ta
80-c
a 20
0
200
400
600
800
Lu
min
esce
nce (
a.u
.)
+/- Controls N/P = 4 N/P = 6 N/P = 10 N/P = 20 N/P = 30 N/P = 40
Nanotechnology for Intracellular Nucleic Acid Delivery
Prof. Ke Zhang
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Delivery peptide nucleic acids (PNAs)
Luciferase splice correction assay
HN
O
PNA
SHN
O
PNAS
R=
Cleavable
Non-cleavable
Mol. Pharm. 2009
Non-cleavable Cleavable
RT-PCR
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An overlooked aspect…
Arrangement of the Nucleic Acids
Physiochemical Properties
of the Carrier
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NaCl
12 h
Thiol-modified oligonucleotide
12 h
High density spherical nucleic acids
Thiol-modified oligonucleotide
12 h
NaCl
12 h
Densely functionalized
(~100 DNA strands/13 nm particle)
Property Conjugate Free nucleic acid
Melting transition
Cooperative andnarrow (~ 2-8 °C)
Broad (~ 20 °C)
StabilityResistance
to nucleasesRapid degradation
Binding Keq = 1.8 x 1014 Keq = 1.8x1012
Lytton-Jean et al. Adv. Mater. 2009, 21, 706-709
Elghanian et al. Science 1997, 1078-1080
Seferos et al. Nano Lett. 2009, 308-311
Lytton-Jean et al. J. Am. Chem. Soc. 2005, 12754-12755
Nanotechnology for Intracellular Nucleic Acid Delivery
Prof. Ke Zhang
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Conjugates are taken up by cells
Giljohann et al. Nano Lett. 2007, 7, 3818-3821
Oligonucleotides per AuNP
Au
NP
s (
x1
05)
pe
r c
ell
BSA-coated AuNPs
Oligo-modifiedAuNP
C166 Endothelial Mouse Cells
Despite being negatively charged, oligo-modified AuNPs
are internalized by C116 cells in high quantities
Cell uptake is dependent on surface DNA density
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Enter all cell lines and primary cells studied
• Breast (SKBR-3, MCF-7, MDA-
MB-231, AU-565, MCF-10A)
• Brain (U87, LN229, U118)
• Bladder (HT-1376, 5637, T24)
• Colon (LS513)
• Cervix (HeLa, SiHa)
• Skin (C166, KB)
• Kidney (MDCK)
• Blood (Sup T1, Jurkat)
• Leukemia (K562)
• Liver (HepG2)
• Kidney (293T)
• Ovary (SKOV-3, CHO)
• Fibroblast (NIH3T3)
• Macrophage (RAW264.7)
Cell lines
Primary cells Epithelial tumor model
15m
• Brain (Rat hippocampus
neurons, astrocytes, glial cells)
• Bladder
• Blood (Mouse erythrocytes,
PBMC, T-cells)
• Pancreas (Mouse beta Islets)
• Skin (Mouse)
Rat hippocampal neurons
SKBR-3 cells
15m
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Targeting cancer cells by antibodies
Anti-her2 antibody
Rapid cell uptake (<4h) >90% knockdown at pM concentrations
JACS 2012
Nanotechnology for Intracellular Nucleic Acid Delivery
Prof. Ke Zhang
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Nucleic acids as transfection agent
• Sharp melting transition
• Nuclease stability
• Increased binding
• High cell uptake
• Gene regulation
?
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Novel chemistry for hollow particle synthesis
KCN
Gold nanoparticles serve as both
the template and the catalyst
JACS 2010
Nanopods
Nanopods from 40 nm templates
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TEM of nanopods from 40 nm templates
Nanotechnology for Intracellular Nucleic Acid Delivery
Prof. Ke Zhang
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22
Hollow spherical nucleic acids
3’ HS-(T-alkyne)10-Binding region- 5’
T-alkyne =
Self-crosslinking, “diblock” oligonucleotide:
JACS 2011
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hSNA MW increases with template size
10 nm template 30 nm template
TEM of hSNAs
Hollow SNAs
10 nm template 30 nm template
TEM of hSNAs
DNA densities are identical
Size 1 10 100 1000 (nm)
Num
ber
%
DLS number-average size distribution
Dh(n) = 30.5 ± 2.5 nm
Dh(n) = 51.5 ± 4.7 nm
hSNA MW increases with template size
Size 1 10 100 1000 (nm)
Num
ber
%
DLS number-average size distribution
Dh(n) = 30.5 ± 2.5 nm
Dh(n) = 51.5 ± 4.7 nm
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hSNAs have sharp melting transitions
Complementary hSNAs hybridize
and form aggregates upon mixing
500 525 550 575 600 625 650
0
200
400
600
800
1000
Norm
aliz
ed
em
issio
n
Emission Wavelength (nm)
Cy3-hSNA
FITC-hSNA Mixture @ RT
Mixture @ 80 oC
FRET signals observed at RT
0 10 20 30 40 50 60 700.0
0.2
0.4
0.6
0.8
1.0
No
rma
lize
d A
bs (
a.u
.)
Temperature (oC)
hSNA (260 nm) Free DNA (260 nm) AuNP-DNA (520 nm)
hSNA produce sharp melting transitions
Nanotechnology for Intracellular Nucleic Acid Delivery
Prof. Ke Zhang
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25
hSNA slow down nuclease degradation
Schematic of nuclease degradation assay
DNA degradation following addition of DNAse I
t½(hSNA) = 4.8 x t½ (free DNA)
0 20 40 60 800
2
4
6
8
10
Free DNA
AuNP-DNA conjugate
hSNA
DN
A d
up
lex
de
gra
de
d (n
M)
Time (min)
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hSNAs enter cells in high quantities
SCC12 cell uptake of Cy5-labeled hSNAs
Oligo-modifiedAuNP
Cy5-labeled hSNA
SCC12 cells incubated with 1 nM Cy5-labeled hSNAs for 24 h
(Right) Cell uptake of DNA-AuNP conjugates and hSNAsquantified by using radiolabeled alkyne-DNA
0
2x106
4x106
6x106
5 nM 10 nM
Nu
mb
er
of
NP
s/c
ell
Incubation concentration
AuNP-DNA
hSNA
1 nM
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Gene regulation control using hSNAs
EGFR mRNA levels EGFR protein levels
Very low cytotoxicity
RNA
DNA
AuNP
KCN
RNA
Chimeric “diblock” oligo
siRNA hSNA
0
20
40
60
80
100
Re
lati
ve
mR
NA
le
ve
l
100 1000 10000
0
25
50
75
100
Lipofectamine 2000
hSNA
Su
rviv
al%
(No
rma
lize
d t
o n
on
-tre
ate
d)
Total DNA (ng)
Nanotechnology for Intracellular Nucleic Acid Delivery
Prof. Ke Zhang
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Acknowledgement
Prof. Karen L. Wooley
Prof. John-Stephen
A. Taylor
Zhou Li
Huafeng Fang
Gang Shen
Yuefei Shen
Prof. Chad A. Mirkin
Joshua I. Cutler
Dan Zheng
Evelyn Auyeung
Liangliang Hao
Jian Zhang
Fei Jia
Alex Lu
Travis Xu
Thank You!
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